title: Cystine dimethylester loading promotes oxidative stress and a reduction in ATP independent of lysosomal cystine accumulation in a human proximal tubular epithelial cell line.
abstract: Using the cystine dimethylester (CDME) loading technique to achieve elevated lysosomal cystine levels, ATP depletion has previously been postulated to be responsible for the renal dysfunction in cystinosis, a genetic disorder characterized by an excessive accumulation of cystine in the lysosomes. However, this is unlikely to be the sole factor responsible for the complexity of cell stress associated with cystinosis. Moreover, CDME has been shown to induce a direct toxic effect on mitochondrial ATP generation. Using a human-derived proximal tubular epithelial cell line, we compared the effects of CDME loading with small interfering RNA-mediated cystinosin, lysosomal cystine transporter (CTNS) gene silencing on glutathione redox status, reactive oxygen species levels, oxidative stress index, antioxidant enzyme activities and ATP generating capacity. The CDME-loaded cells displayed increased total glutathione content, extensive superoxide depletion, augmented oxidative stress index, decreased catalase activity, normal superoxide dismutase activity and compromised ATP generation. In contrast, cells subjected to CTNS gene inhibition demonstrated decreased total glutathione content, increased superoxide levels, unaltered oxidative stress index, unaltered catalase activity, induction of superoxide dismutase activity and normal ATP generation. Our data indicate that many CDME-induced effects are independent of lysosomal cystine accumulation, which further underscores the limited value of CDME loading for studying the pathogenesis of cystinosis. CTNS gene inhibition, which results in intracellular cystine accumulation, is a more realistic approach for investigating biochemical alterations in cystinosis.
authors.affiliation: P. Newsholme: School of Biomedical Sciences, Building 308, Room 122, CHIRI and Faculty of Health Sciences, GPO Box U1987, Curtin University, Perth, WA 6845, Australia. philip.newsholme@curtin.edu.au.
authors.forename: Rodolfo
authors.initials: R
authors.lastname: Sumayao
authors.forename: Bernadette
authors.initials: B
authors.lastname: McEvoy
authors.forename: Natalia
authors.initials: N
authors.lastname: Martin-Martin
authors.forename: Tara
authors.initials: T
authors.lastname: McMorrow
authors.forename: Philip
authors.initials: P
authors.lastname: Newsholme
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D026921:Amino Acid Transport Systems, Neutral / Q000235:genetics; D002374:Catalase / Q000378:metabolism; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D003553:Cystine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D020868:Gene Silencing; D006801:Humans; D007687:Kidney Tubules, Proximal / Q000166:cytology; D008247:Lysosomes / Q000378:metabolism; D018384:Oxidative Stress / Q000187:drug effects; D013482:Superoxide Dismutase / Q000378:metabolism
chemical_list: D026921:Amino Acid Transport Systems, Neutral; C111445:CTNS protein, human; D003553:Cystine; D000255:Adenosine Triphosphate; D002374:Catalase; D013482:Superoxide Dismutase; C034582:cystine dimethyl ester